S. Mailafia1, W. Nafarnda2 and M.Y. Sugun3

1Faculty of Veterinary Medicine, Department of Veterinary Microbiology, University of Abuja, Nigeria.
2Department of Veterinary Public Health and Preventive Medicine, University of Abuja, Nigeria.
3Department of Veterinary Microbiology and Parasitology, University of Maiduguri, Nigeria.

ABSTRACT

The present studyis the first to be conducted in order to determine the occurrence and antimicrobial susceptibility pattern of Aeromonashydrophilastrains from diarrheic patientsattending University of Abuja Teaching Hospital in Nigeria.A total of 153 stool samples were collected from out patients attending University of Abuja Teaching Hospital (UATH). The diarrhoeic patients were screened for the presence of Aeromonashydrophila using routine cultural methods and biochemical characterization. Our results did show that the overall isolation rate is (3. 92%). The prevalence per age group is presented showed that age group 26-30 years having the highest rate of (1.31%) of the total sample analysed. Age groups 11-15 and 16-20 years having the same prevalence rate of  (0.65%) each, while the age groups ≤5, 6-10 and >30 had no prevalence for Aeromonashydrophila. The distribution of A. hydrophilainfection among different sexes has shown that(2.22%) out of the sixA.hydrophila were isolated from diarrheic stools collected from males, while (6.35%) were isolated from females.Statistical analysis showed that X2yates corrected)= 0.05 P= 0.596, OR=1.10 (0.50<OR<2.80).This indicated the level of association between age and rate of Aeromonas infection.Antimicrobial susceptibility patterns of A.hydrophila showed that the isolates were extremely (100%) resistant to Ampicillin, Cephalothin, Gentamicin, Streptomycin, Sulphatriazone, Tetracyclin and Cotrimoxazole. All the isolates are highly susceptible to Colistin and Ceftazidime (100%) followed by Augmentin (83%). They are moderately susceptible to Cefutoxime 50%). Earlier studies revealed resistance to Tetracycline and Cotrimoxazole.Our study confirmed that Aeromonashydrophila as an importantenteropathogenresponsiblefordiarrhoea in humans in Gwagwalada. Diagnostic regime should involve screening of this organism alongside other microorganisms responsible for diarrhoeic symptoms in man and animals.This is the first report to involve Aeromonashydrophila in human diarrhoea and sought regime for choice of antibiotics for the management of the infection.

Keywords: Aeromonas, diarrhoea, antibiotic, susceptibility testing, Occurrence.

INTRODUCTION

Aeromonashydrophilaare Gram-negative, non-spore forming, rod-shaped facultative anaerobic bacilli. They are generally motile by polar flagella (Villariet al., 2003). They grow over a wide range of temperature 0-400C, with human (motile mesophilic) strains growing between 10-400C, with 300C as the optimum temperature, while the non-motile psychrophilic species grow at between 22-280C in soil, food and animal body (Cheesbrough, 2005).  Aeromonaswere classified in the family Vibrionaceae (Jawetzet al., 2004). However, molecular genetic evidence (including 16s rRNAcatalog, 5srRNA sequence, and rRNA-DNA hybridation)suggests they are not closely related to Vibriospecies.In the latest edition of Bergy’s Manual of Systematic Bacteriology; therefore, they are classified as a separate family the Aeromonadaceae(Jawetzet al., 2004). Aeromonasare ubiquitous in fresh and brackish waters. These organisms have also been isolated from a wide variety of sources including soil, sea foods and humans (Michael et al., 2000).

The concentration of Aeromonasvaries with environment in which they are found. In clean rivers, lakes, and storages reservoirs or grocery store products, concentrations are typically around 102 Colony Forming Units (CFU)/ml. The concentration in ground water is generally less than one CFU/ml. Drinking water immediately leaving the treatment plant may contain between 0-102 CFU/ml, withpotentially higher concentration in drinking water distribution systems,thereby attributing it to grow in biofilms (United State Environmental Protection Agency, USEPA, 2005). Higher densities of 108 CFU/ml can be found in waste waters, treated sewage and crude sewage (Holmes et al., 1996). They are also found in sinks, drain pipes, household effluents and are often associated with biofilm. Aeromonashydrophilahave also been isolated from a variety of animal foods, including red meat (beef, pork and veal), poultry and poultry products, fish and shellfish (USEPA, 2005). It has been isolated with variable frequencyfrom different foods being if raw, refrigerated or frozen food of animal origin (Ventura et al., 1998).

Aeromonashydrophila have been implicated in a variety of infections in humans such as gastroenteritis, wound infections (cellulites), septicaemia and diarrhoea. Occasionally,urinary tract infections, meningitis, and peritonitis (Michaelet al., 2000).Aeromonas infections are typically acquired through routes such as ingestion of contaminated water and food, or through contact of the organisms with a break in the skin (Jawetzet al., 2004). They are also implicated in colitis, meningitis, and are frequently isolated from wound infection sustained in aquaticenvironments (Krovaceket al., 1992). They are also being implicated in respiratory infection (Janda and Abbot, 1998). In recent years, Aeromonashydrophilahas gained public health recognition as an emerging pathogen and plays an important role food poisoning associated with human gastroenteritis (Balajiet al., 2004).

The presence of these organisms in human stools is significantly more often associated with diarrhoea than with the carrier state (Kandakai-Olukemiet al., 2007). To the best of ourknowledge, not much study has been carried out in order to ascertain the prevalence of Aeromonashydrophila in outpatients suffering from diarrhoea especially in the areas covered by this study. The lack of defined diagnostic and treatment regimeand definitive control strategy stimulated the antibiotic search involved in this study. The present research therefore is the first to beconducted in order to determine the occurrence and antimicrobial susceptibility pattern of Aeromonashydrophilastrains in diarrheic patientsattending University of Abuja Teaching Hospital in Nigeria.

MATERIALS AND METHODS

Study Area

Federal Capital Territory (FCT), in central Nigeria was created in 1976 from parts of former Nasarawa, Niger, and Kogi States. It situated between latitude 8.250 and 9.200 north of the equator and longitude 6.450 and 7.390 east of Greenwich Meridian and located North of the confluence of rivers Niger and Benue.FCT is bordered by Niger state to the North, Kaduna state to Northwest, Nasarawa to the South-East and Kogi to the West with a land mass of approximately 7,315km2. FCT is currently made up of six Local Government Area Councils namely: Gwagwalada, Kuje, Bwari, Kwali, Abaji and Abuja city situated within the savannah region with moderate climate conditions (Anon, 2012). The study was carried out in University of Abuja Teaching Hospital,Gwagwalada,and Abuja.

Collection of Samples/ Sampling

The authorities of the teaching hospitalswere approached with a letter of introduction seeking itsconsent to undertake the study.The criteria used to  collect the samples after theethical clearance from the Head of Microbiology Department included: consent of the authorities,  availability  of  microbiological  units  in  the  hospital  and  willingness  of  the owners approached to cooperate.

One  hundred and fifty three (153) human stool samples were conveniently collected from the  Teaching Hospital with previous  arrangement made  with  the  doctors  or  matrons  in   hospital  establishment. About 5 gm. or 5 ml of each faecal sample was submitted for the study. The samples  were  collected  when  freshly  voided  faecal  specimens  were  submitted  to  the various laboratories for bacteriological or  parasitological examination.  The samples were scooped  with  a  spatula  and  decanted,  respectively  into  a  labelled  screw  cap  polystyrene bio freeze bottle and tightly screwed.  Care was taken to avoid gross contamination with environmental material.  A corresponding questionnaire was given to the appropriate caregiver.  It  was  used  to  collect  information  on  age,  sex,  type  of  food consumed,  source  of  water  for  domestic  use,  presence  of  vomiting,  fever  and  episodes  of diarrhoea. The periods of sample collection lasted for about four months that is, October 1st2015 to 31st January 2016.   The samples werecollected mostly in the mornings and evenings each day from Mondays through Sundays.

Processing of Samples

The samples were transported to the National Agency for Food and Drug Administration and Control (NAFDAC)reference laboratoryKaduna for examination. The stool samples were picked with the aid of sterilized cotton swabs and weredipped into the prepared tetrathionate broth. It was incubated at 370C for 24 hours. If notused  immediately,  it  was  sent  to  the  Artificial  Insemination  Unit  Laboratory  of  the Nigerian Agricultural  Research and Liaison Service (NEARLS), and was stored in liquid nitrogen (-1960C) until ready for culture and isolation. The conventional culture and isolation method used forthe detection of Aeromonashydrophila as described by Cowan and Steel (1974) were used. This involves  enrichment,  selective  plating,  detection  of  colonies,  preliminary  identification  and complete biochemical identification.

 CultivationandLaboratoryIdentification

The isolation of Aeromonashydrophilawas by the methods adopted byJatau and Yakubu (2004);Jawetzet al., (2004). One gram (1g) of each sample was briefly emulsified in 3 ml of sterile 0.85% (w/v) saline and subsequently vortexed under safety carbine for 30 seconds. Organic debris was allowed to settle down for five minutes. Wet mounts were prepared and examined microscopically with X10 objective followed by X40. Stools with protozoan parasites or worms were excluded from the study. The samples were preenriched in alkaline peptone water (Oxoid, PH 9.0) and sub-cultured after incubation at 370C for 6 hours onto MacConkey agar (Oxiod ) and Sheep blood agar (5% sheep blood) supplemented with 10mg/l ampicillin (SBAA), flowed by incubation at 370C for 24hrs. Ampicillin-resistant β-hemolytic colonies that appeared greyish white, stippled and translucent on SBAA and colonies which failed to ferment lactose on MacConkey agar were Gram stained and Gram negative rods isolated and stored on nutrient agar (Oxoid) slants as presumptive A. hydrophila.Non-lactose fermenting colonies were picked and tested for production ofoxidase. Oxidase positive organisms were purified by re-streaking on nutrient agar slants and pure colonies inoculated onto the surface of prepared nutrient agar slants. They were incubated for 24 hrs at 370C and stored in the refrigerator for further biochemical tests.

 

Biochemical Characterization of the Isolates

Ampicillin-resistant β-haemolytic colonies on SBAA and Non-lactose fermenting colonies on MacConkey agar were subjected to indole, methyl red, Voguesproskauer, citrate IMVIC test, and also inoculated on Kligler Iron Agar (KIA) slants (Oxoids). Those that gave ++++ IMVIC reactions and K/AG (glucose and gas positive, lactose negative) reactions were tested for cytochrome C oxidase activity by Kovac’s method (Cowan and Steel, 1994). Oxidase-positive colonies were examined for amylase activity on Starch-Ampicillin agar (Jatau and Yakubu, 2004). The isolates were further tested for hydrolysis of aesculin and acid production from arabinose The isolates were further tested for resistance to 150µg 0/129 Vibrio static agent (2, 4-diamio-6, 7-diisoprophylpteridine). Owing to the reported increased incidence of pteridineresistant Vibrio cholera, all identified A. hydrophilawere examined for motility in distilled water (Cheesbrough, 2005), and confirmed according to the methods of Cowan and Steel (1994) and McFaddin (2000). The isolates were stored on nutrient agar slants (Oxoid) for further tests.

Antimicrobial Susceptibility Testing

Kirby-Bauer National Committee for Clinical and Laboratory Standard (NCCLS, 2002) modified disc diffusion technique was used to examine the antimicrobial susceptibility of the isolates. The antibiotic multiple disc (AbtekBiologicals Ltd-LotHJ03/P) used comprised of Ampicillin (10µg), Cotrimoxazole (25µg), Gentamicin (10µg), Streptomycin (20µg)Tetracyline (25µg), Cephalothin (5µg), Colistin (25µg), Sulphathiazone (200µg), Cefutoxime (30µg), Ceftazidime (30µg), Augmentin (Amoxycillin and Clauvulanic acid) (30µg).

Each isolate was grown overnight on nutrient agar to obtain isolated colonies. Isolated colonies were transferred to a test tube of sterile saline (0.8% W/V NaCl) and vortexed thoroughly until the turbidity compared to the same with 0.5 McFarland turbidity standards (1x108cells/ml). Within 15 minutes  after standardizing the inoculum, a sterile cotton swab was dipped into the inoculum and excess liquid was removed by pressing the swab firmly against the inside wall of the tube just above the fluid level. The swab was used to streak the entire surface ofMueller –Hinton agar (Oxiod) plates. The plates were allowed to stand for 5 minutes. Antibiotics discs were aseptically placed firmly on the surface of the inoculated agar plates using sterile forceps, and the plates were incubated at 370C for 24 hours. Diameters of zone of inhibition were measured and isolates were characterized as susceptible or resistant according to NCCLS (2002) interpretation chart.

RESULTS

Out of the one hundred and fifty three (153) diarrheic stool samples analysed, 6(3. 92%) were found to be positive for Aeromonashydrophila. The prevalence per age group is presented in Table1. Showed that age group 26-30 years having the highest rate of 2 (1.31%) of the total sample analysed. Age groups 11-15 and 16-20 having the same prevalence rate of 1 (0.65%) each, while the age groups ≤5, 6-10 and >30 had no prevalence for Aeromonashydrophila.The distribution of A. hydrophilainfection among different sexes is shown in Table2. 2 (2.22%) out of the sixA.hydrophila were isolated from diarrheic stools collected from males, while the remaining 4 (6.35%) were isolated from samples collected from females. Table 3 presents the antimicrobial susceptibility patterns of Aeromonashydrophilato eleven antibiotics tested against the isolates. Out of the six Aeromonashydrophilaisolates, two showed varying susceptibility to Cefotaxime and Amoxycillin/ Clauvulanic acid.Colistin, and ceftaxidim, were susceptible to the entire four (6) isolates. However, all the (6) isolates were resistant to cephalothin, Ampicillin, streptomycin, sulphatriazone, tetracycline, and cotrimoxazole.Table 4-5 shows the existence of high level of multiple drug resistance among the strains particularly to cephalothin, streptomycin, sulphatriazone, tetracycline, ampicillin and cotrimoxazole.

Table 1: Prevalence of Aeromonashydrophilain Various Age groups of Diarrhoeic Humans

Age (years)   
No. of

samples  positive

Age (years)

samples  positive

Age (years) percentage positive
≤ 5
84
0
0
6 – 10
10
0
0
11 – 15
6
1
0.65
16 – 20
6
1
0.65
21 – 25
7
0
0
26 – 30
20
2
1.31
> 30
20
2
1.31
Total
153
6
3.92

 

 

 

Sex
No of samples

Collected

No of positive for A.

hydrophila

Percentage

Prevalence

Male
90
 2
2.22
Female
63
 2
6.35
Total
153
6
3.92

Table 2: Prevalence of Aeromonashydrophilainfection in various humansexgroups

X2(yates corrected)= 0.05 P= 0.596, OR=1.10(0.50<OR<2.80)

 

Antibiotics
No of Isolates Susceptible (%)

 

No of Isolates Resistant (%)
Ampicilin
0 (00
6(100)
Cephalothin
0 (00)
6(100)
Colistin
6 (100)
0(00)
Gentamicin
3 (50)
3(50)
Streptomycin
0 (00)
4(100)
Sulphatriazone
0(00)
4(100)
Tetracycline
0(00)
4(100)
Cotrimoxazole
0(00)
4(100)
Ceftazidime
6(100)
0(00)
Cefutoxime
3(50)
3(50)
Augmentin
5(83)
1(17)

Table 3: Antimicrobial susceptibility patterns ofAeromonashydrophila of six isolates

N= 4. N-total number of Aeromonashydrophilatested.

Values in ( ) are percentages

Table4 :AeromonashydrophilaIsolates and Their Resistance Patterns

Isolate                         
No. of brands
Resistance patternsa
% Resistanceb
A. hydrophila
5
AMP,FLX,ERY,PEN
A. hydrophila
6
AMP,FLX,ERY,PEN,CRX
A. hydrophila
6
AMP,FLX,ERY,PEN,CRX,COT
A. hydrophila
4
AMP,FLX,ERY,PEN,CRX,COT
A. hydrophila
3
AMP,FLX,ERY,PEN,CRX,COT,TET
A. hydrophila
3
AMP,FLX,ERY,PEN,CRX,COT
A. hydrophila
2
AMP,FLX,ERY,PEN,CRX
A. hydrophila
1
AMP,FLX,ERY,PEN,CRX,CIT,TET
A. hydrophila
1
AMP,FLX,PEN,CRX,CIT
A. hydrophila
1
AMP,FLX,ERY,PEN,CRX
A. hydrophila
1
AMP,FLX,ERY,PEN,CRX
A. hydrophila
1
AMP,FLX,ERY,PEN,CRX,COT,TET

aResistancepatterns constructed from the antibiogram; antibiotic codes as defined under methodology. bPercentage abstained from the antibiogram

 

Table 5:Percentage resistance of Aeromonashydrophilato different antibiotics

Antibiotic (pg/disk)
No. of strains resistant to antibiotics
%Resistance to antibiotic
Ampicillin (10)
6
100.0
Cephalothin(5)
6
100.0
Colistin(15)
6
00.0
Gentamycin (5)
6
100.0
Streptomycin(30)
6
100.0
Sulphatiazole(25)
6
100.0
Tetracycline (10)
6
100.0
Cotrimoxazole (10)

Ceftazidime (5)

Cefotoxime (10)

Amoxycillin/Clauvulanic acid  (10)

6

6

6

6

100.0

0.00

50.0

17.0

 

DISCUSSION

The recovery of six strains of  Aeromonashydrophilafrom human faecal samples indicates the suitability of this medium for field isolation of members of the genus Aeromonas. The research conducted also documented the occurrence of A.hydrophilain humans in the Gwagwalada area.There are several bases for a diagnosis of aeromoniasis, but the definitive one is isolation of the organism (Nzeakoet al., 2002).  The  identification  of  Aeromonas  strains  from humans  as  revealed  by  this  study  are  therefore  of  public  health significance.  Most  previous  reports  on  the  investigation  of  aeromoniasis  among  human population were based on clinical signs and there have not been much effort to isolate and characterized aeromonae from  human population in Nigeria since reviews of literature from various parts of the country  have revealed that aeromoniasis widely distributed.  There  is  a  pattern  of  low,  medium  and  high prevalence in specific areas and prevalence  also varies among animal species in the same area  (Abbey  et  al.,  2004).  The  confirmatory  diagnosis  of aeromoniasis  is  based on  isolation  and  identification  of the organism  from the  infected host. The isolation and subsequent characterization of Aeromonashydrophila from humans is therefore very significant.

The overall prevalence rate of 3.92 % of Aeromonashydrophilafound inthis study islower than the rate of 10.2 % found in Japan (Arai et al., 1980). It was also  higher  than  the  3.8  %  isolation  rate  reported  by  Kwaga et  al.  (1988)  in Zaria.Conversely, higher prevalence rate of isolation is a signal to public health authorities that aeromoniasis is on the increase. Appropriate measures need to be put in place to control and prevent this disease.    Our results clearly showed theexistence possible seriousness of these disease humans inGwagwalada. The high prevalence of Aeromonasspecies reported in this study warns on public food safety problem in Nigeria.  In  most  parts  of  Nigeria,  people  handle  fish  and  poultry  products with bare hands  and  sometimes  consume contaminated water which may promote the spread of the disease.This  suggests  that  Aeromonashydrophilainfection indicated in our study  might  be  food  borne  or  water  borne  rather  than  air  borne  in  origin.    Otherfactors responsible forthe outbreak may includegeographical location, seasons of the year, health status or hygiene of the individual and methods of isolation.

The distribution of the Aeromonashydrophila from different age groups and sexes   showed that females dissipated higher prevalence (6.35%) than males (2.22%) while the very young age groups had no infection. Age groups of 16-25 years had prevalence of (0.65%) and those within the range age of 26- above 30 years showed prevalence of (0.31%).Statistical analysis showed that X2yates corrected)= 0.05 P= 0.596, OR=1.10 (0.50<OR<2.80).This indicated the level of association between age, sex and rate of Aeromonas infection. The implications of the findings is that Aeromonashydrophila is common in children than adults may be attributed to the fact that children have comparably lower post exposure immunity and are more adventurous than adults. This may expose them to niches and surfaces that are easily contaminated. The habit of indiscriminate picking and putting up objects in the mouth by children also encourages transmission of the infection.

The fact that World Health Organization (WHO) report on infectious diseases recently declared that antibiotic resistance poses a severe threat to human health, and that the problem is growing globally (WHO, 2002). Thus monitoring of antibiotic resistance provides data for antibiotic therapy and resistance control. In addition, selections of antibiotic patterns are sometimes useful as characteristics for species identification, especially for clinical isolates (Jawetzet al., 2007). Antimicrobial susceptibility patterns of A.hydrophila showed that the isolates were extremely (100%) resistant to Ampicillin, Cephalothin,Gentamicine, Streptomycin, Sulphatriazone, Tetracyclin and Cotrimoxazole. All the isolates are highly susceptible to Colistin and Ceftazidime (100%) followed by Augmentin (83%). They are moderately susceptible to Cefutoxime 50%). Earlier studies by Subaskumaret al. (2006) revealed resistance to Tetracycline and Cotrimoxazole and susceptibility forcolistin, gentamicin and ceftazidime. This corroborates well with the findings indicated in our studies. The presence of antibiotic resistant Aeromonashydrophila in diarrhoeic patients in Gwagwalada is of immense public health significance because of the dangers in promoting multiple antibiotic resistances through the colonization of the gastrointestinal tract and conjugal transfer of antibiotic resistance to the normal flora leading to increase in multi- drug antibiotic resistant strains of the bacteria. The apparent resistance of A. hydrophilato antibiotics may be a result of indiscriminate or sub therapeutic use of antibiotics. Multiple drug resistance among Aeromonashyrophilahas been reported from many parts of the world (Sinhaet al., 2004). Multiple drug resistance occurred more in A.hydrophilathan other species of Aeromonasand that isolates from humans and animals could be more resistant to antibiotics (Sinhaet al., 2004). The prevalence of drug resistant strains of Aeromonaspossess great challenge to clinicians and the consumption of animal food and food products like meat, milk and eggs containing this antibiotics may be inappropriate and may require new and mostly expensive antibiotics. Our findings is the first to expose the presence of antibiotic resistant strains of Aeromonashydrophila in diarrhoeic patients with its public health implications and recommends that stringent measures be taken to prevent their occurrence. This study may form basis for Aeromonas research especially in the investigation of risk factors associated with aeromoniasis, search for new drug discovery and control of the disease in humans and animal population and in the environment. On this light, routine diagnostic regime in our hospitals should involve screening of this organism alongside other microorganisms responsible for diarrhoeic symptoms in man and animals.

CONCLUSSION

The objectives of our research have been greatly achieved.  The overall prevalence of A. hydrophila indicated in our study was 3.92% and prevalence was higher for females (6.35%) than males (2.22%). The isolates also developed multi drug resistance to commonly used antimicrobial agents.  In view of the high level of multiple drug resistance observed in our study, regulations should be enforced governing the handling and sales of antibiotics to avoid indiscriminate use of drugs which may enhance the development of resistant mutants. Enlightenment of the public as regards to personal hygiene of individuals, use of antibiotics in animals, water and the environment is highly recommended.

ACKNOWLEDGMENTS

The Authors wish to express their profound gratitude to Mr.P.A. Pam, PrincipalChief Technologist of the Microbiology Department, University of Abuja Teaching Hospital, Gwagwaladaand his team for the technical assistance.

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